Professional Documents
Culture Documents
Topics to Discuss
Introduction: History and definition Process Functions
Aeration Agitation Temperature Control PH control
History
1954 Cell culturing techniques developed 1986 Ortho Biotech's Orthoclone OKT3
kidney transplant rejection first monoclonal antibody treatment
Chiron's Recombivax HB
First genetically engineered human vaccine Approved for the prevention of hepatitis B.
Source: Biotechnology Institute Web Site www.biotechinstitute.org
Bacteria
Robust with strong cell wall Fast growing (20 minute doubling time) High product titer High oxygen demand Fast batch cycle time No viral threat
Bacteria
Products are often intracellular Limited in ability to produce really large molecules (upper limit is probably calcitonin: 45 kDa. Insulin is 6 kDa) Can not produce glycosylated proteins. Can not produce fully humanized antibodies
Aeration System
Sampling System
Harvest System
Operating Modes
Batch Fed Batch Perfusion (continuous feed) Spin filter Centrifuge Settling
Media comparison
Ham's Tissue Culture Medium for Mammalian Cells (amounts dissolved in 1 liter of triple distilled water) L-Arginine L-Histidine L-Lysine L-Methionine L-Phenylalanine L-Tryptophan L-Tyrosine L-Alanine Glycine L-Serine L-Threonine L-Aspartic acid L-Glutamic acid L-Asparagine L-Glutamine L-Isoleucine L-Leucine L-Proline L-Valine L-Cysteine Thiamine hydrochloride Hypoxanthine Folic acid 211 mg 21 mg 29.3 mg 4.48 mg 4.96 mg 0.6 mg 1.81 mg 8.91 mg 7.51 mg 10.5 mg 3.57 mg 13.3 mg 14.7 mg 15 mg 146.2 mg 2.6 mg 13.1 mg 11.5 mg 3.5 mg 31.5 mg 1 mg 4 mg 1.3 mg Biotin Calcium pantothenate Choline chloride i-inositol Niacinamide Pyridoxine hydrochloride Riboflavin Thymidine Cyanocobalamin Sodium pyruvate Lipoic acid CaCl2 MgSO4.7H2O Glucose NaCl KCl Na2HPO4 KH2PO4 Phenol red FeSO4 CuSO4.5H2O ZnSO4.7H2O NaHCO3 0.024 mg 0.7 mg 0.69 mg 0.54 mg 0.6 mg 0.2 mg 0.37 mg 0.7 mg 1.3 mg 110 mg 0.2 mg 44 mg 153 mg 1.1 g 7.4 g 285 mg 290 mg 83 mg 1.2 mg 0.83 mg 0.0025 mg 0.028 mg 1.2 g
(grams/ liter) Minimal Medium for E. coli
Glucose
5g
Na2HPO4
6g
Media Sterilization
Fermentation media can usually be thermally sterilized in the fermentor. Cell culture media is usually filter sterilized into the bioreactor. Thus, fermentors are designed to be sterilized full, and bioreactors are designed to be sterilized empty.
PROCESS FUNCTIONS
Aeration pH Agitation Temperature Control
Aeration Systems
Bioreactors:
Low gas flow rates typically on the order of 0.01 VVM Inlet gas is a mixture of Air (for DO control and CO2 stripping), Oxygen (for DO control without excessive gas flow rates), and CO2 (for pH control. Foaming usually not a problem
Fermentors: High gas flow rates, typically on the order of 11.5 VVM. Inlet gas is primarily air. Occasional applications require oxygen enrichment. Foaming is frequently a problem. Oxygen transfer rate is usually the limitation to productivity.
Oxygen Requirements
0.05 - 0.5 mMoles / L / Hr 150 1500 mg / L/ Hr 0.1 VVM
pH Control
Agitation
Cells are shear sensitive Mixing to prevent gradients in dissolved oxygen and temperature Scale-up based on shear and mixing
Agitator features
Large axial flow impellers Angle mount to eliminate baffles Low RPM / Shear
Scale-up Criteria
Constant Shear
Ss = SL(Dis/ DiL)1/3
Temperature Control
Closed or semi-closed re-circulating temperature control Minimize difference between jacket temperature and bioreactor contents temperature (T<18C) Cascade temperature control
HARDWARE
Seed to Production Tanks Agitators Valves Traps The Specification
Laboratory Bench top equipment for discovery or process development, typically under 30 liters
Pilot Skid mounted equipment for scale up studies, process optimization, or small volume production. Typically under 2000 liters
The Tank
Polished 316L SS pressure vessel. Fully drainable Above 100 liters, designed for complete
CIP
The Agitator
Bioreactor Agitator:
Low shear High mixing capacity Power input typically <1kw/1,000 liters Primary scaling criteria is mixing time.
Fermentor Agitator
High power input Radial impellers (Rushton turbines) are common high speed Power input up to 10 kw/1,000 liters typical Primary scaling criteria is oxygen transfer rate
Bottom Drive
Seal is exposed to direct contact with culture media Shorter shaft Top head is left free for pipe, ports and probes Can open top head on small vessels without removing agitator
Seal Design
Cartridge type double mechanical seal Seals are arranged back-toback. Increasing sealant pressure increases sealing force Sealant is clean steam condensate Seal can be sterilized with vessel, separately, or both. Seal assembly can be pressure tested before installation
Diaphragm Valves
Source: ITT, Pure-Flo, Integrated Block Valve CD, IBV-07(C)
Reference: www.asepco.com
Reference: www.jordanvalve.com
Traps
Fast acting Sanitary Thermostatic Allow adequate drip leg
The Specification
Scope of Work Mechanical Electrical Instrumentation and Controls Testing Requirements Quality Assurance - At Site Options Reference Specifications
Data Sheets
Process and General Data Electrical Requirements Major Equipment Data Piping Control System Instrument Listings
Scope of Work
List all equipment to be included Include items such as tagging, skidding and wiring
Mechanical
Details of equipment Include references to any standard specifications Piping Materials
Electrical
Wire to a common point Reference specifications
Testing requirements
Factory Acceptance Test (FAT) Site Acceptance Test (SAT)
Reference Specifications
Control Systems
Connectors
Millipore Colder
Conventional LSCC
Typical LSCC
Simplified LSCC
Single Use All Biotech Facilities Incorporate Some Single Use Companies Are Developing Processes Using Only Single Use Systems
Bioreactor Size Will Decrease Higher Titers Improved Pharmacology Alternative Expression Systems
Conclusion
Bioreactors need to be specified to meet the specific needs of the cell culture process Conditions of temperature, dissolved oxygen and pH are important to cell growth Hardware and materials need to be chosen that are cleanable and sterilizable The specification should include hardware as well as testing and documentation requirements